The invention generally relates to nucleic acid, and more particularly, to binding of single stranded nucleic acid with biological material of interest in a sample to identify the material.
Nucleic acids such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and protein nucleic acid (PNA) are fundamental components of matter of living organisms. Nucleic acids generally speaking consist of certain constituent parts, or base pairs. The permutations in which these base pairs may be arranged is vast. Nucleic acid sequence analysis, i.e., determining the identity and sequence of the base pairs of a nucleic acid sample, is an important technology. Deciphering nucleic acid sequences is important for disease diagnosis, drug design and understanding of various biological mechanisms. Before 1996, traditional methods laboriously xe2x80x9creadxe2x80x9d the gene sequencing one base pair at a time.
Around 1996, Affymetrix developed a massively parallel sequencing approach, using a DNA chip with which several base pairs can be read simultaneously. A monolayer of specific single stranded DNA (ssDNA) fragments is assembled on an array of pixels (xcx9c1-100 xcexcm2). The type of ssDNA may change from pixel to pixel. These ssDNA fragments act as xe2x80x9cchemical tweezersxe2x80x9d to pick the specific complementary tagged ssDNA from the sample to form double stranded DNA (dsDNA), i.e., a hybridization process occurs. The hybridized region is observed by a fluorescent label applied to the DNA in the sample before exposure to the ssDNA probe fragments. The process is speedier than single-base-pair methods, is specific, can analyze multiple nucleotide sequences, simultaneously can process hundreds of gene sequences and their alterations, and uses advantageously small chips. Practical applications of the parallel sequencing technology include: Affymetrix"" study of p53 gene malfunction (i.e., mutation) responsible for cancer (especially breast cancer); Merck""s study of changes in DNA sequencing as the cell beings to rapidly proliferate (to understand tumor formation); Incyte Pharmaceutical""s disease-specific chips for drug design. Also, massively parallel, quick, sensitive and accurate bio-chip methods may boost the Human Genome Project.
However, Affymetrix"" approach tags unknown to-be-sequenced DNA with a fluorescent dyexe2x80x94altering the sample so that it generally cannot be reused for other tests. Also, because the Affymetrix chip at each pixel has about at least a 5% error margin, the chip includes many repeated pixels, to manage the error margin. The fabrication process for Affymetrix"" chip is expensive, requiring lithographic technique.
DNA sequencing and other protein identification techniques, especially speedy parallel approaches, have important practical applications, such as disease diagnosis, drug design, genetic and cancer screening, deciphering and functional study (such as mutation, gene expression) of genetic code, understanding various biological mechanisms, crime detection, etc. Although advances in DNA sequencing and other protein sequencing technologies have been made in recent years, such procedures still limit and delay workers in the art awaiting sequencing results. Those whose work relies on DNA sequencing and other protein sequencing would be helped by expedited rapidity of sequencing, simplified sequencing, and/or enhanced precision and accuracy. Also, a small portable device useable in a doctor""s office to check, for example, if a patient may eventually develop cancer or how fast the body is likely to break down a specific anti-cancer drug have been generally theorized as of interest. For all these applications, a tool that can perform a nucleic acid analysis of a small size sample for several specific genes (at low concentrations) is highly desirable, but not conventionally provided. Rather, the conventional bio-chip methods undesirably require tagging of the sample plus other disadvantages (such as expensive manufacturing methods, uncertainty in the number of fragments per pixel, etc.).
It therefore is an object of this invention to provide methods and products for detecting the hybridization state of a nucleic acid molecule, without needing to tag the sample. The invention can be used to perform sequence analysis of unknown nucleic acids, such as DNA sequences. Several genes can be probed simultaneously. Sequencing according to the invention is relatively simple and quick, while providing precise and accurate sequencing information. The invention provides a bio-chip and other products for simultaneously analyzing one or more specific nucleic acid fragments (such as genes) in a solution.
In order to accomplish these and other objects of the invention, the present invention in a preferred embodiment provides a tagging-free method to detect binding of molecules, comprising the steps of: (A) providing a sensor comprised of a first layer including a single stranded nucleic acid sequence and a second layer including a photoluminescent material; (B) exposing said sensor to a biological sample for sufficient time for said single stranded nucleic acid sequence to bind to a material of interest in said biological sample; (C) exposing said sensor to light and measuring photoluminescence from said sensor. In a particularly preferred inventive tagging-free method, the measuring step includes sensing photoluminescent light from the second layer when ultraviolet light with wavelength in the range of 200-700 nm is applied to the first layer. In an especially preferred embodiment, the wavelength of the ultraviolet light applied is in the range of 260-265 nm. In an especially preferred embodiment, the first layer is positioned on a first side of the second layer, and said measuring step measures photoluminescence from a second side of said second layer. In a further embodiment, said second side is opposite said first side on said second layer. In another embodiment that is preferred, said first layer is positioned on a first side of said second layer, and said measuring step measures photoluminescence reflected from said first side of said second layer.
In another preferred embodiment, the invention provides a tagging-free sensor comprising a first layer including a single stranded nucleic acid sequence and a second layer including a photoluminescent material.
In another preferred embodiment, the invention provides an apparatus for tagging-free detection of binding of molecules, comprising: a light source; a sensor having a nucleic acid layer and a photoluminescent layer; and a photoluminescence detector. In especially preferred embodiments, the light source is an ultraviolet light source, an infrared light source, or a visible light source. In other especially preferred embodiments, the detector is a light detector in the infrared to ultraviolet range. Where an ultraviolet light source is used, the ultraviolet light source in a particularly preferred embodiment provides ultraviolet light at a range of about 260-265 nm.
In another preferred embodiment, the invention provides a method of making a tagging-free sensor, comprising: contacting a single stranded nucleic acid sequence with a photoluminescent material. A particularly preferred embodiment of such an inventive method includes depositing photoluminescent material on a substrate to form a surface, and thereafter modifying the surface by ion exchange treatment with a metal salt, followed by ion-embedding, followed by exposing the ion-embedded material to reactive media to form photoluminescent particles.
In the above-mentioned methods, products and apparatuses, a particularly preferred embodiment of the invention uses DNA, RNA and/or PNA as the single stranded nucleic acid. In an especially preferred embodiment, the first layer comprises an ssDNA monolayer. In a particularly preferred embodiment, the sensor comprises ssDNA as said first layer grafted onto the second layer.
In a preferred embodiment, the nucleic acid sequence is between 5 and 200 base pairs. In an especially preferred embodiment, the sequence is about 25 base pairs. Another particularly preferred embodiment provides a discontinuous first layer comprising different nucleic acid sequences in different sections of said first layer.
Further, another preferred embodiment of the invention provides a tagging-free method to detect binding of antigens, comprising the steps of: (A) providing a sensor comprised of a first layer including an antibody and a second layer including a photoluminescent material; (B) exposing said sensor to a biological sample for sufficient time for said antibody to bind to an antigen of interest in said biological sample; (C) measuring photoluminescence from said sensor. In a particularly preferred embodiment, the first layer is discontinuous and comprises different antibodies.
In another preferred embodiment, the invention provides an apparatus for tagging-free detection of antibody binding, comprising: a light source; a sensor having a first layer including an antibody and a second photoluminescent layer; and a light detector.
Additionally, another preferred embodiment of the invention provides a tagging-free sensor comprising a first layer including an antibody and a second layer including a photoluminescent material. In a particularly preferred embodiment, the first layer is discontinuous and comprises different antibodies. In another especially preferred embodiment, different known antibodies are included.
Further detailed embodiments of the above-mentioned inventive methods, products and apparatuses are as follows.
Another particularly preferred embodiment of the invention in the second layer uses aromatic polymers, doped or undoped metal oxides, sulfides, selenides, arsenides, tellurides, and/or nitride and phosphide nanocomposites. In another preferred embodiment, the second layer may comprise a matrix material, with said photoluminescent material associated with said matrix material. In a further preferred embodiment, said photoluminescent material is embedded in said matrix material. In an especially preferred embodiment, the second layer comprises polystyrene. In an especially preferred embodiment, the second layer comprises photoluminescent particles in a polymer matrix. In a further preferred embodiment, the photoluminescent particles may be doped or undoped compounds selected from the group consisting of group II and group VI. In another preferred embodiment, as such a compound is used doped or undoped zinc sulfide. Another especially preferred embodiment is one in which the second layer comprises a nanocomposite. In a further preferred embodiment, the second layer comprises a thin-film or a support. In another preferred embodiment, the second layer comprises a polymer. In another preferred embodiment, the second layer has fluorescence when excited by light with a wavelength in the 200-700 nm range.